Dopamine transporters (DATs) are critical for controlling local extracellular concentrations of dopamine (DA) and thereby DA synaptic transmission in the CNS. The number of DAT molecules expressed at the neuronal surface determines the rate of extracellular DA clearance. Rapid changes in surface expression of DAT are determined by endocytic trafficking, i.e., the equilibrium between internalization into endosomes and recycling from endosomes back to the cell surface. Clathrin-mediated internalization is the major rate-limiting step regulating endocytic cycling of DAT. However, the molecular mechanisms that control DAT internalization through coated pits are unknown. Moreover, whereas studies of DAT endocytosis have used model expression systems, it remains to be established whether similar mechanisms mediate internalization of DAT in neurons. Our long-term goal is to understand regulation of DAT trafficking in the CNS. The focus of this proposal is on elucidation of the basic molecular machinery and protein-protein interactions that occur during endocytic trafficking of DAT and on specific regulatory mechanisms of these interactions leading to dynamic changes in surface expression of DAT and DA transport. Heterologous expression systems will be used to develop tools and approaches that will then be applied to rat nigrostriatal DA neurons. In Aim #1, the hypothesis that DAT internalization depends on the interaction of specific sequences in DAT with the clathrin adaptor AP2 complex will be tested by defining the molecular mechanisms and functional role of DAT-AP2 interactions. A combination of molecular, biophysical and biochemical techniques will be utilized. In Aims #2 and #3, the hypothesis that signaling through presynaptic receptors and protein kinases regulates endocytosis of DAT will be tested. In particular, the role of tyrosine phosphorylation and DA D2 receptor (D2R) activation in regulating the endocytosis and/or recycling of DAT and in controlling the number of DATs expressed on the cell surface will be analyzed. Understanding the molecular mechanisms involved in DAT trafficking will provide new insights into the regulation of DAT activity and thereby DA neurotransmission in the brain, as well as into the actions of DA and D2R drugs.